Not applicable.
The invention relates to methods for producing transgenic plant cells and plants with modified 5-aminolevulinic acid biosynthesis, to the use of nucleic acid molecules encoding a protein which act as a 5-aminolevulinic acid synthase (ALAS) for producing transgenic plant cells and plants, methods for identifying effectors, and the use of effectors, of aminolevulinic acid biosynthesis, in particular the use of herbicidally active inhibitors of plant 5-aminolevulinic acid biosynthesis in crops, preferably in transgenic crops.
Herbicides are used widely in modern agriculture for controlling undesired vegetation on agricultural land. Despite the use of herbicides, an efficient control of weeds and harmful plants is not always possible in a satisfactory manner since highly effective herbicides which have a broad spectrum of action are frequently not tolerated by useful plants, or else the development of resistance phenomena can be observed.
So far, it has been attempted to circumvent these disadvantages by useful plants which tolerate herbicides with a non-selective action. The herbicide tolerance can be generated by modifying the plant enzyme which is inhibited by the selected herbicide in such a way that it is less sensitive to the herbicidally active substance. For example, WO 95/3459 describes plants which express genes of protoporphyrinogen oxidase variants and thus have an increased tolerance to herbicidal diphenyl ethers and other inhibitors of plant protoporphyrinogen oxidase.
Herbicide tolerance was also achieved by introducing, by genetic engineering, enzymes into the metabolism of crop plants which are capable of deactivating the herbicidally active substance applied (for example EP-A-0 242 236; EP-A-0 343 100).
It is known that the biosynthesis of 5-aminolevulinic acid (ALA) is a rate-determining and regulatory metabolic step for porphyrin synthesis in plants, animals, fungi and bacteria. In plants and in most prokaryotes, with the exception of the alpha group of the purple bacteria, ALA is produced from glutamate via the so-called C5 metabolic pathway. The C5 metabolic pathway (C5 pathway) consists of three reaction steps which are catalyzed by the enzymes glutamyl-tRNA synthetase (EC 6.1.1.17), glutamyl-tRNA reductase (EC 1.2.1.-.) and glutamate-1-semialdehyde aminotransferase (GSAAT, EC 5.4.3.8.) and, in plants, is localized in the plastids.
GSAAT has been isolated from a variety of organisms, and the structural genes of the enzyme have been cloned for example from the plants Arabidopsis thaliana, tobacco, barley and soybean.
Plant GSAAT can be produced by recombinant technology by means of heterologous expression of its cDNA (Berry-Lowe et al. (1992) Plant Physiol. 99: 1597-1603).
GSAAT is inhibited by 3-amino-2,3-dihydrobenzoic acid. After application of 3-amino-2,3-dihydrobenzoic acid, the treated plants are incapable of synthesizing ALAs and thus no longer the porphyrin compounds chlorophyll, heme and siroheme (Beale (1990) Plant Physiology 93: 1273-1279). Such plants develop highly chlorotic tissue which is destroyed when exposed to light.
An alternative biosynthetic pathway for the production of ALA exists in animals, fungi, yeasts and the purple bacteria of the alpha group, for example Rhodobacter sphaeroides or Rhodobacter capsulatus. In this synthetic pathway, which is termed Shemin metabolic pathway, succinyl-CoA and glycine are condensed in a single reaction step which is catalyzed by the enzyme 5-aminolevulinic acid synthase (ALAS, EC 2.3.1.37.), to give ALA (Kikuchi et al. (1958) Journal of Biological Chemistry 233: 1214-1219). In eukaryotes, the reaction steps of the Shemin metabolic pathway proceed in the mitochondria.
ALAS has been isolated from a number of organisms. The ALAS structural gene has been isolated, inter alia, from Saccharomyces cerevisiae, Rhodobacter sphaeroides, Rhodobacter capsulatus, humans and mice.
ALAS, being the first enzyme of the Shemin tetrapyrrole biosynthesis pathway, has already been examined early for its regulation at the transcriptional level and at the enzyme activity level (Lascelles (1968) Biochem. Soc. Symp. 28: 49-59, Gamick et al. (1975) Journal of Biological Chemistry 250: 9215-9225). It has been found in particular in the case of prokaryotic 5-aminolevulinate synthases that heme is an important direct feedback regulator of enzyme activity.
It was an object to provide a resistance principle to plant 5-amino acid levulinic acid synthesis inhibitors which is suitable under practice conditions and to provide a rational method for finding such inhibitors which is suitable for practice conditions.
The introduction of a heterologous ALAS activity in tobacco plants using the cDNA of Saccharomyces cerevisiae ALAS (SEQ ID NO: 4) has already been described by Zavgorodnyaya et al. (1997, Plant Journal 12, 169-178).
However, the ALA synthesis rate in the plant must be sufficient to ensure the ALA requirement for the physiologically necessary porphyrin biosynthesis. On the other hand, ALA production must not be so high as to damage the plant directly or indirectly. This is why the C5 biosynthesis pathway for ALA is regulated in plants.
Similarly, it must be guaranteed that the ALA synthesis in the transgenic plants which contain a heterologous ALAS activity does not have detrimental consequences for the plants.
Surprisingly, it has now been found that plants can be produced which are resistant to herbicidally active inhibitors of the C5 metabolic pathway, for example to GSAAT inhibitors, when certain heterologous ALAS genes are expressed, in useful plants, in substitutive or complementary fashion so that non-selectively acting inhibitors of the C5 metabolic pathway can be employed in said transgenic crops of useful plants as herbicides.
The present invention therefore provides a method for producing transgenic plants with modified 5-aminolevulinic acid biosynthesis in which the metabolic pathway which is inhibited specifically by a herbicide is circumvented by a heterologous gene expression which complements or substitutes the inhibition. For a suitable complementation or substitution, the plants are equipped, by means of one or more heterologous nucleic acid molecules, with one or more additional or alternative biosynthetic steps which lead to the end product of the inhibited metabolic pathway so that a resistance of the transgenic plant to the herbicidally active inhibitor of the C5 metabolic pathway results.
FIG. 1 depicts lasmid p35StpASN.
FIG. 2 depicts lasmid pcva13.
FIG. 3 depicts lasmid pALAS3f.vec.